As was discussed in Promoter characterization part, MerR protein, in the form of homodimer, binds to a dyad sequence between the -10 element and -35 region of element PmerT. The binding, however, is independent of the presence of Hg (II). When the apo-MerR dimer binds to the dyad symmetrical operator DNA between the -35 and – 10 elements of mercury inducible promoter, PmerT, which has an unusually long spacer of 19 bp, the contact of RNA polymerase is sloppy. When Hg (II) is available in the environment, ions bind to the interface of MerR dimer. The Hg-bound MerR dimer can result in a structural distortion of PmerT, allowing the RNA polymerase tight contacts to be made, leading to the expression of down-stream genes (Fig.1).

'''Fig 1. The concentration of Hg-bound dimers can be ‘diluted’ by Hg-free dimers. Because both of them can bind to PmerT promoter but only Hg-bound dimer activates the transcription, the activation effect of MerR is therefore determined by the concentration of Hg (II), the binding affinity to PmerT (namely the sequence of MerR binding site) and the concentration of Hg-free MerR dimer (or the total concentration of MerR). '''

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The model shown in Fig 1 indicates that the apo-merR and Hg-bound merR have a competition. Because both of them can bind to promoter but only Hg-bound dimer are capable of transcription activation, the regulation role of MerR is therefore determined by the concentration of Hg (II), the binding affinity to PmerT (namely the sequence of MerR binding site) and the expression intensity of MerR. We speculate that except manipulation at the dyad sequence of PmerT, the threshold of MerR response can be also manipulated by controlling the concentration of MerR in cytosol.

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As with the bacteria in natural environment, the concentration of MerR is stabilized at a certain level. In order to verify the speculation, promoters from partsregistry constitutive promoter library with different strength were prefixed before BBa_B0034+MerR coding sequence to exogenously maintain MerR expression at different intensity (Fig 2). Tthe sensitivity of PmerT under different MerR concentrations can be denoted by mercury threshold concentration at which reporter (GFP) expression emerges. Furthermore, we used pSB1A2 and pSB3K3 as backbones because of their different copy numbers which could also result in different MerR expression intensity (Fig 3), which was confirmed by an additional experiment.

'''Fig 2. The construction of the system used for MerR operation characterization.''' Promoters from the partsregistry with different intensities were selected, leading to varying MerR concentrations. The transcription of PmerT is controlled by the percentage of Hg-bound MerR dimer, thus the expression of GFP can reflex the concentration of Hg (II).

'''Fig 4. Dose response behavior of each constitutive-promoter-controlling MerR. A: Pc-MerR was cloned into pSB1A2 backbone and PmerT-BBa_E0840 into pSB3K3 backbone. It can be observed that GFP expression level drop dramatically at high Hg (II) concentration levels. From the top to the bottom, they are J23101, J23103, J23108, J23109, J23112, J23114, J23116, and J23117. B: Pc-MerR was cloned into pSB3K3 backbone and PmerT-GFP into pSB1A2 backbone. Data were processed as the same with Fig.4-A. '''

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As was mentioned above, MerR dimers act as a repressor of PmerT or an activator in the presence of Hg (II). A weaker constitutive promoter leads to the less production of MerR, raising the proportion of Hg-bound MerR dimer, resulting in a higher threshold (Fig 4, Fig 5). On the other side, data of characterization demonstrated that GFP intensity, which is measured by microplate-reader, needed higher Hg (II) concentration to rise to a considerable level when the constitutive promoter is strong (Fig 4, Fig 5).

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When the Hg (II) concentration became as high as 10^-5M, cells can not tolerate the toxicity (Fig.4). However, data fit Hill function well apart from this. The ‘n’ values ranged from 2~4, determined by in silico fit of origin data.

'''Fig 5. The expression intensity of MerR significantly determines the threshold of sensitivity to mercury (II). '''Five representative lines are selected and it can be seen that the thresholds have varied apparently. The letter in the bracket after the promoter name denotes the backbone (pSB3K3 or pSB1A2) where Pc-RBS-merR was cloned. The deeper the colour, the stronger the expression level of MerR is, leading to a higher threshold.

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In summary, promoters from partsregistry constitutive promoter library with different strength were prefixed before MerR coding sequence to exogenously maintain MerR expression at different intensity. The sensitivity of PmerT under different MerR concentrations can be denoted by mercury threshold concentration at which reporter (GFP) expression emerges. Data demonstrates that cells with different MerR intensity exhibited correspondingly different sensitivity to mercury, indicating that the stronger the expression intensity of MerR is, the higher threshold will be represented.

As was discussed in Promoter characterization part, MerR protein, in the form of homodimer, binds to a dyad sequence between the -10 element and -35 region of element PmerT. The binding, however, is independent of the presence of Hg (II). When the apo-MerR dimer binds to the dyad symmetrical operator DNA between the -35 and – 10 elements of mercury inducible promoter, PmerT, which has an unusually long spacer of 19 bp, the contact of RNA polymerase is sloppy. When Hg (II) is available in the environment, ions bind to the interface of MerR dimer. The Hg-bound MerR dimer can result in a structural distortion of PmerT, allowing the RNA polymerase tight contacts to be made, leading to the expression of down-stream genes (Fig.1).

Fig 1. The concentration of Hg-bound dimers can be ‘diluted’ by Hg-free dimers. Because both of them can bind to PmerT promoter but only Hg-bound dimer activates the transcription, the activation effect of MerR is therefore determined by the concentration of Hg (II), the binding affinity to PmerT (namely the sequence of MerR binding site) and the concentration of Hg-free MerR dimer (or the total concentration of MerR).

The model shown in Fig 1 indicates that the apo-merR and Hg-bound merR have a competition. Because both of them can bind to promoter but only Hg-bound dimer are capable of transcription activation, the regulation role of MerR is therefore determined by the concentration of Hg (II), the binding affinity to PmerT (namely the sequence of MerR binding site) and the expression intensity of MerR. We speculate that except manipulation at the dyad sequence of PmerT, the threshold of MerR response can be also manipulated by controlling the concentration of MerR in cytosol.

As with the bacteria in natural environment, the concentration of MerR is stabilized at a certain level. In order to verify the speculation, promoters from partsregistry constitutive promoter library with different strength were prefixed before BBa_B0034+MerR coding sequence to exogenously maintain MerR expression at different intensity (Fig 2). Tthe sensitivity of PmerT under different MerR concentrations can be denoted by mercury threshold concentration at which reporter (GFP) expression emerges. Furthermore, we used pSB1A2 and pSB3K3 as backbones because of their different copy numbers which could also result in different MerR expression intensity (Fig 3), which was confirmed by an additional experiment.

Fig 2. The construction of the system used for MerR operation characterization. Promoters from the partsregistry with different intensities were selected, leading to varying MerR concentrations. The transcription of PmerT is controlled by the percentage of Hg-bound MerR dimer, thus the expression of GFP can reflex the concentration of Hg (II).

Fig 3. The strategy of backbones swiching. We used pSB3K3, a low-copy number backbone, and pSB1A2, high-copy, to differ the expression of merR.

We carefully tested every combination (the strength of constitutive promoter controlling MerR expression and the backbone copy number) under induction with a Hg (II) concentration gradient ranging from 10^-9M to 10^-5M. The concentration gradient of Hg (II) is: 0, 1E-9, 3E-9, 5E-9, 8E-9, 1E-8, 3E-8, 5E-8, 8E-8, 1E-7, 3E-7, 5E-7, 8E-7, 1E-6, 3E-6, 5E-6, 8E-6, 1E-5, 3E-5, and 5E-5. Cell culture was cultivated for 8 hours and then dilute with fresh LB in a ratio of 1:100 and continue to incubate until the OD600 reached 0.4~0.6.

Fig 4. Dose response behavior of each constitutive-promoter-controlling MerR. A: Pc-MerR was cloned into pSB1A2 backbone and PmerT-BBa_E0840 into pSB3K3 backbone. It can be observed that GFP expression level drop dramatically at high Hg (II) concentration levels. From the top to the bottom, they are J23101, J23103, J23108, J23109, J23112, J23114, J23116, and J23117. B: Pc-MerR was cloned into pSB3K3 backbone and PmerT-GFP into pSB1A2 backbone. Data were processed as the same with Fig.4-A.

As was mentioned above, MerR dimers act as a repressor of PmerT or an activator in the presence of Hg (II). A weaker constitutive promoter leads to the less production of MerR, raising the proportion of Hg-bound MerR dimer, resulting in a higher threshold (Fig 4, Fig 5). On the other side, data of characterization demonstrated that GFP intensity, which is measured by microplate-reader, needed higher Hg (II) concentration to rise to a considerable level when the constitutive promoter is strong (Fig 4, Fig 5).

When the Hg (II) concentration became as high as 10^-5M, cells can not tolerate the toxicity (Fig.4). However, data fit Hill function well apart from this. The ‘n’ values ranged from 2~4, determined by in silico fit of origin data.

Fig 5. The expression intensity of MerR significantly determines the threshold of sensitivity to mercury (II). Five representative lines are selected and it can be seen that the thresholds have varied apparently. The letter in the bracket after the promoter name denotes the backbone (pSB3K3 or pSB1A2) where Pc-RBS-merR was cloned. The deeper the colour, the stronger the expression level of MerR is, leading to a higher threshold.

In summary, promoters from partsregistry constitutive promoter library with different strength were prefixed before MerR coding sequence to exogenously maintain MerR expression at different intensity. The sensitivity of PmerT under different MerR concentrations can be denoted by mercury threshold concentration at which reporter (GFP) expression emerges. Data demonstrates that cells with different MerR intensity exhibited correspondingly different sensitivity to mercury, indicating that the stronger the expression intensity of MerR is, the higher threshold will be represented.